Structure of Lactose Synthase (LS): In order to understand the molecular basis of interaction between b-1,4-galactosyltransferase (Gal-T1) and a-Lactalbumin (LA), and their interactions with the sugar donor, acceptor and metal ions, and the mechanism of modulation of oligosaccharide acceptor specificity of Gal-T by LA, our primary goal has been to determine the three dimensional structure of Gal-T1-LA complex (LS) with various ligands. The crystal structures of LS bound with various substrates, solved at 2 A resolution (1) show that Gal-T1 upon ligand binding undergoes a conformational change that positions the residues in such a way that it creates binding sites for a metal ion, for the sugar acceptor molecule, and exposes the LA binding site to facilitate its binding to Gal-T1. Although the structure of Gal-T1 was determined by a group of scientists in France (Gastinel et al.) about a year and a half ago, the structure did not explain many of the mutational data previously published from various laboratories, including ours. The crystal structure determined at 2 A resolution in our lab showed that the overall conformation of the catalytic domain of Gal-T1 is very similar to the structure determined by the French group, except for the region comprising residues 345 to 365 and the side chain orientation of Trp314. This large (about 20 A ) conformational change repositions the residues creating sugar acceptor and metal ion binding sites and exposes the LA binding site. Upon ligand binding the side chain of Trp314 moves towards the inside of the catalytic pocket and the residue His347 is positioned in such a way that it can partake in coordinating Mn2+ion with Met344, and Asp254 of D252VD254 sequence. Site directed-mutagenesis and kinetic analysis, and the crystal structures of the mutants show that Asp254 and His347 strongly bind metal ligand, while Met344 coordinates less strongly and can be substituted by serine, glutamine or alanine. The sugar acceptor binding site of Gal-T1 is a hydrophobic pocket, formed by residues Arg359, Phe360 and Ile363, where the N-acetyl group of GlcNAc binds. In the Glc bound structure of Gal-T1-LA complex, the hydrophobic pocket is blocked by the reorientation of the side chain of Arg359, which maximizes the interactions with Glc. LA holds the Glc molecule in the acceptor-binding site by hydrogen bonding with its O-1 hydroxyl group, where as the hydrophobic patch of LA interacts with the exposed hydrophobic region of Gal-T1. Thus, the conformational change in the region comprising the residue 345 to 365 of Gal-T1, is a prerequisite for the metal ion, sugar acceptor and LA binding, and for catalysis. We propose that the structure obtained by the French group represents the inactive state of the enzyme (conformation-I) that upon substrate binding undergoes a large conformational change (conformation-II) creating sugar acceptor binding site. This explains previous mutational and enzyme kinetics data of Gal-T1. The crystal structure of Gal-T1 with less preferred sugar donor substrate, UDP-Glc reveals the role of LA in glucosyltransferase activity of Gal-T1. The transfer of Glc from UDP-Glucose (UDP-Glc) to N-acetylglucosamine by Gal-T1 is very poor, but it is stimulated 30-fold by LA. The crystal structure of Gal-T1-LA complex with bound UDP-Glc and Mn2+ reveals that the role of LA in stimulating the glucosyltransferase activity of Gal-T1 is at the enzyme kinetic mechanism level and not at the level of molecular interactions between UDP-Glc and Gal-T1. The kinetic data suggests that the binding of LA stabilizes the enzyme UDP-Glc complex in the conformation II. For stearic reasons after LA binding the reverse conformational change i.e. from II to I is not possible unless the product N-acetyl-cellabiosamine is formed. This conformational change from I to II is important not only for the LA and acceptor binding but also for the donors that have weak affinity for the enzyme. Based on the structural details of the complexes of Gal-T1 with less preferred sugar-nucleotide donor substrates e.g UDP-GalNAc, we have rationally designed a novel glycosyltransferase with dual transferase activities. By a single point mutation in the sugar-nucleotide binding pocket of Gal-T1 we have successfully converted Gal-T1 into an enzyme that is equally efficient as b1-4-N-acetyl-galactosaminyltransferase. Ramakrishnan,B and Qasba, PK. J.Mol.Biol. 2001;310,205-218. Ramakrishnan,B , Shah PS and Qasba PK. J. Biol.Chem. 2001, WEB SIte August 2 (in press). Z01 BC 09304-05